Extended Toolboxes Enable Efficient Biosynthesis of Multiple Products from CO₂ in Fast-Growing <i>Synechococcus</i> sp. PCC 11901

Cyanobacteria are known to be photoautotrophic cell factories capable of converting CO2 into valuable chemicals. The newly discovered marine cyanobacterium Synechococcus sp. PCC 11901 (hereafter PCC 11901) offers several advantages like rapid growth, high biomass, and high salinity tolerance, representing a promising chassis. To promote its application, we developed genetic toolboxes applicable to PCC 11901 in this study. First, a cobalamin (VB12)-independent chassis was constructed, allowing for cheaper cultivation. Second, genome copy numbers and transformation methods were, respectively, measured and optimized. Then, 14 neutral sites were identified and characterized within the genome PCC 11901, providing locations for genetic integration of exogenous cassettes. Subsequently, promoter libraries were developed, reaching an expression range of approximately 800 folds for constitutive promoters and an induction fold of up to approximately 400 for inducible promotors, respectively. As a proof of concept, natural production of the total lipid and phycocyanin was investigated using VB12-independent chassis, which realized an increase of 14.91% with lipid content compared with that of the wild-type strain. Further, we engineered the synthetic pathways of glucosylglycerol (GG) into PCC 11901 using the established toolboxes, reaching 590.41 ± 21.48 mg/L for GG production and self-sedimentation in photoreactors with the highest OD750 nm at 17.57 ± 0.77. Finally, the GG-producing strain grew well in seawater, reaching 324.50 ± 5.34 mg/L in shaking flask, which provided new strategies for cyanobacteria cultivation and production. Our work here made it possible to develop the fast-growing PCC 11901 as efficient carbon-neutral cell factory in the future.